Sex Differences in the Neurotoxic Effects of Adenosine A₁ Receptor Antagonism During Ethanol Withdrawal: Reversal With an A₁ Receptor Agonist or an NMDA Receptor Antagonist

Abstract

Background

Neuronal adaptations that occur during chronic ethanol (EtOH) exposure have been observed to sensitize the brain to excitotoxic insult during withdrawal. The adenosine receptor system warrants further examination in this regard, as recent evidence has implicated adenosine receptor involvement in the behavioral effects of both EtOH exposure and withdrawal.

Methods

The current studies examined effects of adenosine A₁ receptor manipulation on neuronal injury in EtOH-naïve and EtOH-withdrawn male and female rat hippocampal slice cultures. EtOH-naïve and EtOH pretreated (43.1 to 26.9 mM from days 5 to 15 DIV) cultures were exposed to the A₁ receptor agonist 2-Chloro-N⁶-cyclopentyladenosine (CCPA; 10 nM), the A₁ receptor antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX;10 nM), or the N-methyl-D-aspartate (NMDA) receptor antagonist D,L,-2-amino-5-phosphovalerate (APV; 20 μM) at 15 days in vitro (DIV). Cytotoxicity was measured in the primary neuronal layers of the dentate gyrus, CA3 and CA1 hippocampal regions by quantification of propidium iodide (PI) fluorescence after 24 hours. Immunohistochemical analysis of A₁ receptor abundance was conducted in EtOH-naïve and EtOH pretreated slice cultures at 15 DIV.

Results

Twenty-four hour exposure to DPCPX in EtOH-naïve slice cultures did not produced neurotoxicity in any region of slice cultures. Though withdrawal from 10 day EtOH exposure produced no toxicity in either male or female slice cultures, exposure to DPCPX during 24 hours of EtOH withdrawal produced a marked increase in PI uptake in all hippocampal culture subregions in female cultures (to ~160% of control values). A significant effect for sex was observed in the CA1 region such that toxicity in females cultures exposed to the A₁ antagonist during withdrawal was greater than that observed in male cultures. These effects of DPCPX in EtOH withdrawn female and male slices were prevented by co-exposure to either the A₁ agonist CCPA or the NMDA receptor antagonist APV for 24 hours. No differences in the abundance of A₁ receptors were observed in male and female EtOH-naïve or EtOH pretreated cultures.

Conclusions

The current findings suggest that the female hippocampus possesses an innate sensitivity to effects of EtOH exposure and withdrawal on neuronal excitability that is independent of hormonal influences. Further, this sex difference is not related to effects of EtOH exposure on A₁ receptor abundance, but likely reflects increased NMDA receptor-mediated signaling down-stream of A₁ inhibition in females.

The 2004 to 2005 Substance Abuse and Mental Health Services Administration (SAMHSA) National Survey of Drug Use and Health reported lifetime alcohol use among persons aged 12 and older at 82.9%, with past year use prevalence estimated at 66.5%. Binge alcohol use was reported by 22.7% of adults, and heavy alcohol use was reported by 6.6% of adults (Substance Abuse and Mental Health Services Administration, 2006). These data are reflective of the 2001 to 2002 National Epidemiologic Survey on Alcohol and Related Conditions (NESARC), in which 8.5% of adults in the United States reported a past year alcohol use disorder. Of those individuals, 3.8% reported alcohol dependence (Falk et al., 2006; Grant et al., 2004). Alcohol dependence poses serious consequences for an individual, as neuronal and cognitive abnormalities have been noted in detoxified alcoholics (Chanraud et al., 2007; Tedstone and Coyle, 2004; Vik et al., 2004). Much research has been devoted to elucidating adaptive changes in expression and/or sensitivity of receptors and ion channels that occur during long-term alcohol use. Namely, following chronic alcohol use, in vivo and in vitro animal models have demonstrated up-regulation and increased sensitivity of N-methyl-D-aspartate (NMDA) receptors (Hu and Ticku, 1995; Kalluri et al., 1998; Prendergast et al., 2000a) and voltage-sensitive calcium channels (Harris, 1999; Little, 1991; Watson and Little, 1999), down-regulation of γ-amino butyric acid type-A (GABA_A) receptors (Devaud et al., 1997; Mahtre and Ticku, 1994; Mahtre et al., 1993), potentiation of serotonin type-3 (5-HT₃) receptors (in McBride et al., 2004), and inhibition or stimulation of 5′-triphosphate-gated purinergic (P2X) receptors (Davies et al., 2006). It has been postulated that these adaptive neuronal changes that occur during chronic ethanol exposure predispose the brain for excitotoxic insult during ethanol withdrawal (Hoffman et al., 1990; Hunt, 1983; Littleton, 1998; Prendergast et al., 2004) and contribute to alcohol craving (De Witte, 2004; Koob, 2003).

To date, research on adenosine receptor involvement during excitotoxicity has been primarily limited to models of ischemia and hypoxia (reviewed by de Mendonca et al., 2000). Recently, however, adaptive changes induced by ethanol exposure and withdrawal have been noted in neuronal adenosine receptors. Four adenosine receptor subtypes (A₁, A_2A, A_2B, A₃) have been characterized in mammalian tissue (Fredholm et al., 1994, 2001). In the rat CNS, cell surface A₁ receptors are most abundant, exhibiting the greatest density in the hippocampus, cortex, cerebellum, thalamus, brainstem, spinal cord, and basal ganglia (Reppert et al., 1991; Rivkees et al., 1995). They are tightly coupled with G_i/o proteins (Fredholm et al., 1994; Nanoff et al., 1995) and A₁ agonism may influence numerous intracellular effecter systems. Inhibitory signaling mechanisms have been described in several different models, including Chinese hamster ovary cells, smooth muscle, and neuronal cells. These mechanisms include inhibition of adenylyl cyclase (Akbar et al., 1994; Freund et al., 1994; Van Calker et al., 1979) causing a subsequent reduction in cyclic adenosine monophosphate accumulation (Akbar et al., 1994; Peakman and Hill, 1996; Van Calker et al., 1978) and inhibition of P/Q- and N-type calcium channels (Ambrosio et al., 1997; Gundlfinger et al., 2007). A₁ agonism may also increase potassium conductance (Li and Henry, 1992; Segal, 1982), and activate phospholipases A₂, C, and D (Akbar et al., 1994; Gerwins and Fredholm, 1995; Rogel et al., 2005, 2006). Consequently, A₁ receptor activation results in neuronal inhibition via postsynaptic hyperpolarization (Li and Henry, 1992; Segal, 1982) and presynaptic inhibition of neurotransmitter release (Dunwiddie and Haas, 1985; Fredholm and Dunwiddie, 1988; Prince and Stevens, 1992).

Recent in vivo behavioral data has also provided evidence for A₁ receptor mediation of ethanol withdrawal behavior in rodents. Concas et al. (1996) demonstrated that withdrawal from ethanol dependence induced by intragastric intubation (4 times daily for 6 days) produced increased [³H]2-Chloro-N⁶-cyclopentyladenosine ([³H]CCPA) binding in homogenized rat cerebellar cortical membranes at 3, 12, and 24 hours of withdrawal. Likewise, Jarvis and Becker (1998) demonstrated increased binding of the selective A₁ agonists [³H]CCPA and N⁶- cyclohexyladenosine ([³H]CHA) in homogenized mice cerebral cortex at 8 hours of withdrawal following a single or multiple withdrawal episode(s). Further, administration of the selective A₁ agonist CCPA produced and anxiolytic effect during withdrawal in these mice, and pretreatment with the selective A₁ receptor antagonist 1,3-Di-propyl-8-cyclopentylxanthine (DPCPX) reversed the CCPA effect (Prediger et al., 2006). Additionally, microinjections of the A₁ agonist 2-chloroadenosine (2-CADO) into the central nucleus of the inferior colliculus at 19 hours of ethanol withdrawal has demonstrated a trend toward reduction in clonus during audiogenic seizures in rats (Feng and Faingold, 2000). Little is known, however, regarding the role that A₁ receptors may have in mediating ethanol or ethanol withdrawal-induced neuronal injury.

The current series of studies were designed to examine effects of adenosine A₁ receptor agonism and antagonism alone and in combination on neuronal viability following 24 hour drug exposure in ethanol-naïve and ethano pre-exposed organotypic hippocampal slice cultures. Further studies were conducted to assess the effects of co-exposure to an NMDA receptor antagonist and an A₁ receptor antagonist on neuronal viability. It was hypothesized that A₁ antagonism would produce excitotoxic neuronal death in hippocampi undergoing ethanol withdrawal. We hypothesized that con- current exposure the A₁ agonist and antagonist or concurrent exposure to the NMDA antagonist and A₁ antagonist would attenuate the hypothesized excitotoxicity produced by A₁ antagonism alone.

EXPERIMENTAL PROCEDURES

Organotypic Hippocampal Slice Culture Preparation

Eight-day old male and female Sprague-Dawley rat pups were humanely euthanatized (Harlan Laboratories; Indianapolis, IN) prior to aseptic whole brain removal. Brains were immediately transferred into chilled dissecting medium (4°C) made of Minimum Essential Medium (MEM), 25 mM HEPES, 2 mM glutamine, and 50 μM streptomycin/penicillin (adapted from Stoppini et al., 1991). Bilateral hippocampi were removed, cleaned of extra tissue under a dissecting microscope, and placed into room-temperature culture medium, composed of dissecting medium with the addition of 36 mM glucose, 25% (v/v) Hanks’ balanced salt solution, 25% heat-inactivated horse serum (HIHS), and 0.05% Penicillin/Streptomycin. Hippocampi were then coronally sectioned at 200 μm using the McIllwain Tissue Chopper (Mickle Laboratory Engineering Co. Ltd., Gomshall, UK) and placed into fresh culture medium. Intact slices were selected and placed onto Millicell-CM 0.4 μm biopore membrane inserts preincubated in 1 ml of culture medium at 37°C in a 35 mm 6 well culture plate. Three slices were placed on each insert, yielding 18 slices per plate. Excess medium from the top of the membrane insert was aspirated to allow the slices exposure to the incubator atmosphere of 5% CO₂/95% air. Slices were allowed 5 days to attach to the insert membrane before any experiments were conducted. Culture medium supplies were obtained from Gibco BRL (Gaithersburg, MD), with the exception of HIHS (Sigma-Aldrich Co., St. Louis, MO). Care of animals was carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23), as well as the University of Kentucky’s Institutional Animal Care and Use Committee.

CCPA, DPCPX, and APV Exposure in Ethanol-Naïve Cultures

At 5 days in vitro (DIV) all cultures were transferred into fresh preincubated culture medium in new 6 well culture plates. At 10 DIV cultures were transferred into new culture plates with refreshed medium. Control cultures aged to 15 DIV was again put into fresh culture medium and the remaining cultures were exposed to 10 nM CCPA (Sigma), 10 nM DPCPX (Sigma), 20 μM APV (Sigma), DPCPCX + CCPA, or DPCPX + APV in culture medium. DPCPX stock was dissolved in DMSO before the addition of culture medium, yielding a DMSO concentration of 0.01%. Additional control slices were exposed to medium with 0.01% DMSO. A₁ agonist and antagonist drugs were chosen based on their A₁ selectivity, as CCPA reportedly has a 10,000-fold selectivity for A₁ receptors (Klotz et al., 1989; Lohse et al., 1988) and DPCPX has the highest reported A₁ antagonist value at 740-fold selectivity (Bruns et al., 1987; Lohse et al., 1987). Further, experimental concentrations of the A₁ receptor ligands were chosen to be near reported dissociation constants (K_d) at the A₁ receptor, those being 2.9 nM for CCPA in cultured rat fetal forebrain (Nicolas and Daval, 1993) and 3.3 nM for DPCPX in the presence of endogenous adenosine in hippocampal slices (Alzheimer et al., 1991). A relatively low concentration of the specific NMDA receptor antagonist APV was chosen (20 μM) based on cell culture and electrophysiology models that have typically reported significant inhibition of NMDA receptor activity at APV concentrations ranging from 5 to 100 μM (e.g. Tu et al., 2007; Xu et al., 2005). All culture medium contained the fluorescent nucleic acid stain propidium iodide (PI) which penetrates dying or compromised cells with damaged membranes at a concentration of 3.74 μM (Zimmer et al., 2000). Uptake of PI was quantified 24 hours after the onset of drug exposure (16 DIV).

Ethanol Exposure and Withdrawal

At 5 DIV, additional cultures were randomly transferred to new plates containing either 1 ml of standard culture medium (control) or 50 mM ethanol in culture medium. All plates were then transferred to topless polypropylene containers containing 50 ml of double-distilled water for control plates and 50 mM ethanol in double-distilled water for ethanol-treated plates. Containers were then placed into sealable plastic bags and filled to capacity with 5% CO₂/95% air before being returned to the incubator in an effort to reduce ethanol evaporation. The same treatment was repeated at 10 DIV. At 15 DIV, all cultures were removed from culture medium containing ethanol and placed into fresh culture medium to begin a 24 hour withdrawal period.

Previous work employing this model has demonstrated a 50% or greater decline in ethanol concentration over 5 days when beginning with an ethanol concentration calculated at 100 mM (Prendergast et al., 2004). To examine possible loss of ethanol, aliquots of cell culture medium containing ethanol were taken at 5 DIV (when ethanol was first administered at a calculated starting concentration of 50 mM), 10 DIV to assess the decrease in ethanol concentration due to evaporation and/or metabolism over 5 days, and again at 15 DIV to assess the ethanol concentration in the medium before drug exposure. After sampling and before analysis all aliquots were stored at −80°C. Ethanol concentration was obtained by measurement of alcohol oxidase activity through oxygen utilization detected by a Clark-type amperometric oxygen electrode on an Analox ethanol analyzer (Analox, London, UK).

CCPA, DPCPX, and APV Treatment During Ethanol Withdrawal

At 15 DIV, all plates were removed from their polypropylene containers. Any cultures previously treated with ethanol were put into 1 ml of ethanol-free culture medium to begin 24-hour withdrawal, with the addition of 10 nM CCPA, 10 nM DPCPX, 20 μM APV, DPCPX + CCPA, or DPCPX + APV, or no drug in culture medium. Fluorescent images were taken 24 hours after drug administration. All experiments were replicated 5 to 7 times with mean N values of approximately 18 slices/group.

Immunohistochemistry

An immunohistochemical assay of A₁ receptor abundance was conducted with additional hippocampal slice cultures in male and female control and ethanol-exposed (10 day, not withdrawn) at 15 DIV. Cultures were washed twice in 0.9% phosphate-buffered saline (PBS) and fixed for 30 minutes in 10% formalin. Cultures were then washed twice in PBS and incubated for 45 minutes in PBS buffer with the addition of 0.1% Triton-X and 0.005% bovine serum albumin to permeabilize cell membranes. Cultures were then transferred into fresh culture plates and incubated at 4°C in permeabilization buffer with the addition of the monoclonal primary antibody (1/200 dilution) against the C-terminal of the rat adenosine A₁ receptor for 24 hours (Sigma). Following the 24 hour incubation with primary antibody, cultures were washed twice with PBS and incubated with permeabilization buffer with the addition of fluorescein isothiocyanate (FITC)-conjugated secondary antibody (sheep antirabbit; 1/200 dilution) for 24 hours at 4°C (Sigma). Fluorescent microscopy following 24 hour incubation with FITC was used to quantify A₁ receptor immunoreactivity in the granule cell layer of the dentate gyrus (DG) and the pyramidal cell layers of the cornu ammonis 3 (CA3) and CA1 regions. Background due to nonspecific FITC fluorescence was measured using the method described above with the omission of the primary antibody.

Fluorescent Microscopy

Cytotoxicity, as measured by PI uptake, and A₁ receptor immunoreactivity, as measured by FITC fluorescence, was visualized via fluorescent microscopy. Images were taken using SPOT Advanced version 4.0.2 software for Windows (W. Nuhsbaum Inc., McHenry, IL) with a 5× objective on an inverted Leica DMIRB microscope (W. Nuhsbaum Inc.) fitted for fluorescence detection (mercury-arc lamp) and connected to a personal computer via a SPOT 7.2 color mosaic camera (W. Nuhsbaum Inc.). Propidium iodide has a maximum excitation wavelength of 536 nm and was excited using a band-pass filter that excites the wavelengths between 515 and 560 nm. The emission of PI in the visual range is 620 nm. The secondary antibody, conjugated with FITC, was excited using a band-pass filter at 495 nm (520 nm emission). Fluorescent intensity (arbitrary optical units) was analyzed by densitometry using Image J (National Institutes of Health, Bethesda, MD) in the granule cell layer of the DG and the pyramidal cell layers of the CA3 and CA1 regions of the hippocampus. For each PI stained slice, background fluorescent intensity was subtracted from each region’s measurement prior to statistical analysis. Before analyzing A₁ immunoreacitivity, nonspecific FITC fluorescence from control and ethanol exposed cultures was subtracted from each region.

Statistical Analysis

No differences were present in PI fluorescence between control values and DMSO-control treated values, and therefore the 2 groups were combined for all analyses. A two-way analysis of variance (ANOVA) was conducted (treatment × sex) within each hippocampal region (CA1, CA3, DG). Previous work using this organotypic slice culture model has demonstrated increased susceptibility to insult in the CA1 region during ethanol withdrawal as compared to the CA3 and DG regions, therefore it was hypothesized that the greatest toxicity, as measured by PI uptake, would occur in the CA1 region (Prendergast et al., 2004; Self et al., 2005). When appropriate, Fisher’s LSD post hoc analyses were interpreted. The significance level was set at p < 0.05. Before graphing, all fluorescent intensity measurements were converted to percent control values.

RESULTS

Effects of CCPA & DPCPX on Neurotoxicity in Ethanol Naïve Cultures

Initial studies were conducted to assess the effects of 24 hour exposure to the selective A₁ receptor agonist CCPA (10 nM) or the selective A₁ receptor antagonist DPCPX (10 nM) at 16 DIV on neuronal injury. Neither CCPA nor DPCPX produced significantly greater PI uptake compared to control cultures in the primary cell layer of any hippocampal region. Additionally, co-exposure to CCPA and DPCPX produced no toxicity in any region (Table 1).

Table 1.

Propidium Iodide Uptake in Male and Female Ethanol-Naïve Hippocampal Cultures Exposed to CCPA (10 nM) and/or DPCPX (10 nM)

	CA1	CA3	DG
CCPA
Female	102.0 (4.6)	95.3 (6.6)	90.5 (5.5)
Male	101.3 (3.3)	101.7 (5.0)	103.1 (3.1)
DPCPX
Female	99.4 (2.4)	95.6 (2.8)	98.4 (3.5)
Male	108.2 (3.8)	97.3 (3.0)	99.7 (3.6)
CCPA + DPCPX
Female	103.7 (4.3)	109.2 (5.0)	105.0 (4.8)
Male	104.8 (4.3)	100.6 (5.4)	103.4 (4.2)

Neither CCPA and/or DPCPX produced toxicity significantly greater than control cultures in males or females. All data are presented as percent control values.

Effects of CCPA and/or DPCPX on Neurotoxicity in Ethanol-Withdrawn Cultures

The decline in ethanol concentration in cell culture medium was measured as the difference between the starting concentration and the concentration 5 days after initial ethanol exposure. At 5 DIV, the calculated starting concentration of ethanol (50 mM) yielded an actual ethanol concentration of 43.1 mM. At 10 DIV, the concentration had decreased to 26.9 mM, yielding a concentration approximately 46% below the initial calculated value (50 mM), despite measures taken to minimize evaporation. At 15 DIV, as cultures were removed from ethanol to begin a 24 hour withdrawal period, the ethanol concentration was 29.0 mM (Table 2). Twenty-four hours of withdrawal from 10 day continuous ethanol exposure (calculated starting concentration of 50 mM) did not produce significantly increased PI uptake compared to control cultures in any hippocampal region in either female or male cultures.

Table 2.

Ethanol Concentration in Culture Medium at 5, 10, and 15 DIV

DIV	5	10	15
mg/dl	198.39	123.93	133.50
mM	43.1	26.9	29.0

The calculated starting concentration of ethanol was 50 mM. ethanol in culture medium was refreshed at 5 DIV and 10 DIV. 24 hours of withdrawal begin at 15 DIV.

Further studies were conducted to assess the effects of A₁ agonism (CCPA; 10 nM) and antagonism (DPCPX; 10 nM) on neurotoxicity during ethanol withdrawal. Cultures exposed to CCPA at the onset of 24 hour ethanol withdrawal did not show significant differences in PI uptake compared to control cultures for either sex, in any hippocampal region. In contrast, a sex × treatment interaction in the CA1 region [F(7,768) = 6.028, p < 0.001] indicated that DPCPX exposure for the duration of the 24 hour ethanol withdrawal period produced a marked increase in toxicity in female cultures, but not male cultures (Fisher’s LSD post hoc, p < 0.001). This marked toxicity in female cultures was approximately 30% greater than that observed in male cultures. Additionally, post hoc comparisons within each sex revealed that in female ethanol-withdrawn cultures, DPCPX produced significant toxicity as compared to ethanol-naïve DPCPX-exposed cultures and ethanol-withdrawn control cultures (Fisher’s LSD post hoc, p < 0.001 for female cultures and p < 0.05 for male cultures; Fig. 1). In female cultures co-exposed to DPCPX and CCPA for the duration of the 24 hour ethanol withdrawal period, DPCPX-induced toxicity was significantly attenuated in the CA1 region (Fisher’s LSD post hoc, p < 0.001). In the DG, a main effect of treatment was present [F(7,790) = 6.846, p < 0.001], such that DPCPX exposure for the duration of ethanol withdrawal produced significantly greater toxicity than DPCPX exposure in ethanol-naïve cultures and ethanol-withdrawn control cultures (Fisher’s LSD post hoc, p < 0.01). Further, co-exposure to DPCPX and CCPA significantly reduced DPCPX-induced toxicity (Fisher’s LSD post hoc, p < 0.001). Similarly, in the CA3 region a main effect of treatment was present [F(7,789) = 6.612, p < 0.001], such that DPCPX exposure for the duration of 24 hour ethanol withdrawal produced toxicity significantly greater compared to DPCPX exposure in ethanol-naïve cultures and ethanol-withdrawn control cultures (Fisher’s LSD post hoc, p < 0.001). Again, co-exposure to DPCPX and CCPA significantly reduced DPCPX-induced toxicity (Fisher’s LSD post hoc, p < 0.001). Despite the lack of a statistical interaction in the DG or CA3 region, it is likely that the treatment effect is attributable to the difference in mean PI uptake between female DPCPX-treated cultures and ethanol withdrawn control cultures, as females showed a trend for greater PI uptake compared to male cultures (Fig. 1). Representative images of hippocampal slices are presented in Fig. 3.

Fig. 1.

Effects of exposure to the A1 antagonist DPCPX or co-exposure to DPCPX and the A₁ agonist CCPA on neurotoxicity in male and female organotypic hippocampal slice cultures during 24 hours of withdrawal from 10 days of ethanol exposure (43.1 to 26.9 mM). Exposure to DPCPX during ethanol withdrawal (EWD) produced significant sex-dependent toxicity in CA1 pyramidal cells of female cultures and sex-independent toxicity CA3 pyramidal cells and dentate granule cells, effects that were prevented by co-exposure to CCPA. *p < 0.01 versus female controls and ethanol withdrawn slices; **p < 0.01 versus all other groups, including male cultures treated with DPCPX during ethanol withdrawal; #p < 0.05 versus females cultures exposed to DPCPX during ethanol withdrawal. ##p < 0.05 versus EWD+DPCPX.

Fig. 3.

Representative images of propidium iodide fluorescence in ethanol-naïve and ethanol-withdrawn organotypic hippocampal slice cultures exposed to CCPA and/or DPCPX. (A) Female control; (B) female ethanol withdrawn; (C) female ethanol withdrawn + DPCPX; (D) female ethanol withdrawn + CCPA + DPCPX; (E) female ethanol withdrawn + CCPA + APV.

Effects of Co-Exposure to DPCPX & APV on Neurotoxicity in Ethanol Withdrawn Cultures

Further studies were conducted to determine whether NMDA receptors mediated the observed toxicity produced by A₁ receptor antagonism during ethanol withdrawal. Cultures were co-exposed to DPCPX (10 nM) and the selective NMDA receptor antagonist APV (20 μM) for the duration of the 24 hour ethanol withdrawal period. A two-way (sex × treatment) ANOVA revealed a significant interaction within the CA1 region [F(3,151) = 2.90, p < 0.05]. Post hoc comparisons revealed a sex difference in this region for cultures exposed to DPCPX for the duration of the ethanol withdrawal period, such that females had significantly greater PI uptake than male cultures (Fisher’s LSD post hoc; p < 0.001). The DPCPX-induced toxicity observed within female cultures was significantly attenuated by co-exposure to APV (Fisher’s LSD post hoc; p < 0.001). As in previous studies, main effects for treatment were observed in the dentate gyrus [F(3,151) = 3.56, p < 0.05] and CA3 regions [F(3,147) = 6.26, p < 0.001]. Post hoc analysis indicated that, in both hippocampal regions, DPCPX exposure during 24 hour of ethanol withdrawal modestly, but significantly increased PI uptake, effects that were significantly attenuated by co-exposure to APV (Fig. 2). Exposure to APV did not alter PI uptake in any other groups. Representative images of PI uptake in ethanol withdrawn slice cultures are shown in Fig. 3.

Fig. 2.

Effects of exposure to the A₁ antagonist DPCPX and the NMDA receptor antagonist APV on neurotoxicity in male and female organotypic hippocampal slice cultures during 24 hours of withdrawal (EWD) from 10 days of ethanol exposure (43.1 to 26.9 mM). Exposure to DPCPX during ethanol withdrawal produced significant sex-dependent toxicity in the CA1 pyramidal cells, an effect that was prevented by co-exposure to APV. *p < 0.01 versus controls and ethanol withdrawn slices; **p < 0.05 versus all other groups including male cultures exposed to DPCPX during withdrawal. #p < 0.05 versus females cultures exposed to DPCPX during ethanol withdrawal.

Immunohistochemistry: A₁ Receptor Immunoreactivity

Immunoreactivity was quantified in male and female control and 10 day ethanol exposed (not withdrawn) cultures, both at 15 DIV. A one-way ANOVA comparing male and female A₁ immunoreactivity in control slices revealed no sex difference. To compare ethanol and control cultures for each sex a two-way ANOVA (sex × treatment) was conducted, but found no sex difference or treatment difference in any hippocampal region. Due to the lack of a sex difference in either control or ethanol cultures, males and females were combined to test for a difference in A₁ immunoreactivity between control and ethanol cultures independent of sex. The analysis revealed that 10 day ethanol exposure did not produce a significant change in A₁ receptor immunoreactivity, in any region, as compared to control cultures of the same DIV (Table 3).

Table 3.

A₁ Receptor Immunoreactivity in Ethanol-Exposed Male and Female Cultures

	CA1	CA3	DG
Female cultures	108.6 (4.7)	103.4 (4.9)	110.4 (6.2)
Male cultures	106.8 (6.7)	100.6 (6.7)	110.5 (7.8)

No significant changes in A₁ receptor abundance were observed. Values are presented as percent control.

DISCUSSION

A wealth of data have demonstrated that acute and possibly protracted ethanol withdrawal is a period of heightened neuronal excitability in humans, animals, and in vitro culture systems. This neuronal excitability is hypothesized to reflect adaptive changes in the function and/or expression of multiple neuronal and/or glial proteins, which likely contribute to withdrawal behaviors and alcohol craving. Receptor systems and ion channels mediating ethanol’s effects include NMDA receptors, GABA_A receptors, L-type calcium channels, 5-HT₃ receptors, nicotinic acetylcholine receptors, and P2X receptors, as changes in function and/or density of these proteins have been noted in both human studies (Dillon et al., 1991; Freund and Anderson, 1996) and animal models, as noted above. Recent work suggests that both ethanol exposure and withdrawal in rodents influences extracellular adenosine accumulation and adenosine receptor signaling. Acute ethanol exposure produces an increase in adenosine release in rat cerebellar synaptomsomes (Clark and Dar, 1989a) and adenosine accumulation in NG108-15 cells and S49 lymphoma cells (Nagy et al., 1989). Extracellular accumulation of adenosine is partly due to inhibition of adenosine uptake by the equilibrative nucleoside transporter type 1, as exposure to ethanol concentrations of 2.5 to 100 mM has been shown to produce a 12 to 15% decrease in [³H]adenosine uptake in rat cerebellar synaptosomes (Clark and Dar, 1989b). Ethanol causes changes in A₁ receptor density, though it is unclear whether this is caused by chronic ethanol exposure or withdrawal. An up-regulation of A₁ receptors has been noted after chronic ethanol exposure (Daly et al., 1994) and at 3 hours following the last ethanol administration while rats were still intoxicated (Concas et al., 1996). Also, increased A₁ density has been observed during withdrawal 8 to 24 hours following the last ethanol administration with no change in agonist binding affinity (Concas et al., 1996; Jarvis and Becker, 1998). As recently mounting in vivo evidence suggests adenosine receptor involvement in ethanol withdrawal behaviors (Batista et al., 2005; Connole et al., 2004; Dar, 1990, 2006; Dar and Clark, 1992; Dar et al., 1983; Feng and Faingold, 2000; Kaplan et al., 1999; Prediger et al., 2004; Prediger et al., 2006), the present experiments were designed to examine the effect of adenosine A₁ receptor agonism and antagonism during ethanol withdrawal on neuronal injury in cultured male and female hippocampi. In the rat and human hippocampus, A₁ receptors are most dense in the CA1 region (Fastbom et al., 1987; Svenningsson et al., 1997). Further, previous literature has noted increased sensitivity of CA1 pyramidal cells during ethanol withdrawal to different neurotoxic agents (Prendergast et al., 2000a,b, 2001, 2004; Self et al., 2005; Wilkins et al., 2006), suggesting that A₁ receptor manipulation may cause the greatest effect in the CA1 region.

Withdrawal from 10 days of exposure to ethanol (ranging from 43.1 to 26.9 mM) did not produce overt toxicity in any region of slice cultures, as measured by PI uptake. It must be noted however, that examination of other neuronal markers may reveal less overt neuronal compromise following ethanol exposure and withdrawal (after Wilkins et al., 2006). This ethanol exposure regimen was chosen for its pharmacological relevance, as the declining ethanol concentration over the 5 day treatment period produced ethanol levels in culture medium from 198.39 mg/dl to 123.93 mg/dl. Neither exposure to DPCPX nor CCPA altered neuronal viability in ethanol-naïve slice cultures. However, 24 hours of exposure to DPCPX in ethanol pre-exposed cultures produced marked neurotoxicity. Granule cells of the dentate gyrus, as well as pyramidal cells in both the CA3 and CA1 regions, of female cultures demonstrated markedly increased toxicity in response to A₁ receptor antagonism during ethanol withdrawal. Conversely, DPCPX exposure during ethanol withdrawal appeared to have little effect in male cultures in these regions. Co-exposure to the A₁ agonist CCPA with DPCPX was able to attenuate all DPCPX-induced toxicity in ethanol-with-drawing cultures, demonstrating the A₁ receptor selectivity of the observed DPCPX effects.

The increased neurotoxicity in DPCPX-exposed female cultures undergoing ethanol withdrawal, as compared to male cultures, suggests the existence of an innate sex difference in sensitivity to effects of ethanol on A₁ receptor signaling or on signaling downstream of A₁ receptor activity, such as the NMDA receptor system as suggested by our finding that APV blocked DPCPX potentiation of ethanol withdrawal. Though very little work has examined sex differences in adenosine signaling or receptor density, 1 study did report a sex difference in A_2A receptor ligand binding in the frontoparietal cortex of juvenile, but not adult, rats (Shirayama et al., 2001), Similarly, adult male and female CD-1 mice display similar mRNA levels for hippocampal A₁ receptors (von Arnim et al., 2002). In the current studies, immunohistochemical identification of A₁ receptor abundance suggested that the increased toxicity observed in females was not related to ethanol-induced changes in A₁ receptor density, again, suggesting that the key ethanol-induced changes may lie downstream of A₁ signaling.

A less specific female vulnerability to neurotoxic insult during ethanol withdrawal may have also contributed to the potentiated DPCPX-exposure response in female cultures during ethanol withdrawal. Previous reports have suggested that the female brain of both humans and rodents is more vulnerable to neurotoxic insult during chronic alcohol use and subsequent withdrawal (e.g. Hashimoto and Wiren, 2008; Hommer et al., 1996, 2001; Prendergast, 2004; Prendergast et al., 2000b). Past literature has suggested that sex differences in human responses to acute alcohol are mainly due to differences in body lipid/water ratio (Marshall et al., 1983) and ethanol metabolism (Frezza et al., 1990). The current data, however, indicate that innate sex differences in neuronal function exist in response to ethanol exposure that are independent of hormonal influence, as peripheral influences are abolished at postnatal day 8 with use of this cell culture model. Behaviorally, sex differences in ethanol withdrawal induced seizures have also been reported. Male mice experience increased seizure severity upon repeated ethanol withdrawal, but females show no sensitization of seizure susceptibility, though seizure propensity was the same in initial withdrawal episodes (Veatch et al., 2007). Further, signs of ethanol withdrawal are abolished more quickly in female rats, though females are slower to develop ethanol dependence (Alele and Devaud, 2007; Devaud and Chadda, 2001; reviewed in Devaud et al., 2003).

Adenosine receptors likely interact both pre- and postsynaptically with receptors and/or ion channels to mediate ethanol’s effects. Presynaptically, A₁ receptor manipulation affects neurotransmitter release, such that A₁ agonism with CCPA reduces and A₁ antagonism with DPCPX amplifies the release of excitatory amino acids in rat hippocampal slices (Di Iorio et al., 1996). Specifically, activation of A₁ receptors decreases glutamatergic neurotransmission (Clark and Dar, 1989c; Lopes et al., 2002; O’Kane and Stone, 1998) and reduces NMDA-induced cell death (Finn et al., 1991). Further, it has been reported using homogenated fetal and adult guinea pig hippocampal slices that pretreatment with CPT (an A₁ antagonist) blocked ethanol-induced inhibition of K⁺-stimulated glutamate release. Conversely, exposure to CCPA and exogenous adenosine inhibited K⁺-stimulated glutamate release at a magnitude similar to ethanol (Reynolds and Brien, 1995). During ethanol withdrawal, NMDA receptors are up-regulated as a consequence of chronic ethanol exposure (Harris et al., 2003), increasing susceptibility to excitotoxicity. In the current studies, the combination of A₁ antagonism with DPCPX (i.e. increased glutamate release) and increased NMDA receptor density provides a mechanism for significantly increased neurotoxicity during withdrawal, as observed in the female hippocampal cultures. Findings demonstrating that APV blocked the potentiating effects of DPCPX strongly support this argument and are consistent with a prior report (Prendergast et al., 2000a) demonstrating that female hippocampi were more sensitive than male hippocampi to the excitatory effects of the NMDA receptor modulating polyamine spermidine following ethanol pre-exposure, than were male hippocampi. The effect of A₁ agonism using CCPA (i.e. decreased glutamate transmission, decreased NMDA receptor function, neuronal inhibition) in the presence of DPCPX during ethanol withdrawal was likely sufficient to block DPCPX-initiated glutamate-induced neurotoxicity at up-regulated NMDA receptors. Postsynaptically, NMDA and A₁ receptors also interact to alter excitatory currents. In rat hippocampal neurons, A₁ activation has been shown to inhibit NMDA receptor-mediated currents (de Mendonca and Ribeiro, 1993; de Mendonça et al., 1995). Additonally, A₁ receptors co-localize with the NMDA receptors subunits NR1, NR2A, and NR2B in the postsynaptic density (Rebola et al., 2003). Thus, manipulation of the adenosinergic receptor system has the potential to mediate ethanol withdrawal-induced neurotoxicity presynaptically by inhibiting glutamate release and subsequent action on NMDA receptors, or postsynaptically by altering NMDA receptor function.

The current data support an innate sex difference in A₁ receptor signaling, such that antagonism during ethanol withdrawal caused greater NMDA receptor-dependent neurotoxic damage in females compared to males. Future studies will need to focus on the adult hippocampus to begin expanding on how adenosine receptor signaling mediates NMDA receptor signaling to produce ethanol withdrawal effects.